During pregnancy three generations of DNA co-exist. Mum's, baby's and the germ line of baby. During the first and second trimester the majority of methylated cytosines from the DNA of baby's progenitor germ line cells, called primordial germ cells (PGCs) are removed. This act is essential to remove errors in methylation acquired during gametogenesis in the parents, and/or during early development of baby after fertilization. If errors in cytosine methylation are not removed, the abnormally methylated alleles have a risk of being inherited as disease epialleles in the following generation. Given that the environment can stably influence the genome, including the genome of baby's PGCs in utero, there is a need to understand the mechanisms that regulate DNA demethylation in PGCs in order to develop strategies to guard against the transmission of disease epialleles in future generations. Recently my group discovered that DNA demethylation in human PGCs is regulated in two phases however the mechanisms underlying demethylation globally (phase 1) and locally (phase 2) are unclear. In this project we aim to uncover new details on the dynamics of DNA demethylation in human PGCs and use conditional deletions of mouse PGCs in vivo as well as differentiation of mouse PGCs from embryonic stem cells in vitro to address hypotheses regarding the specific mechanisms responsible for demethylation in the mammalian germ line. In aim 1 we will identify the dynamic removal of cytosine methylation at base resolution for the very first time in human PGCs and address the hypothesis that Ubiquitin- like, containing PHD and RING finger domains, 1 (Uhrf1) repression by protein arginine methyltransferase 5 (Prmt5) is responsible for the phase 1 DNA demethylation in mammals. In aim 2 using a conditional deletion in mouse PGCs we will address the hypothesis that Dnmt1 maintains cytosine methylation at discreet loci in PGCs in the absence of its major cofactor Uhrf1. In aim 3, we turn to phase 2 demethylation to directly address the hypothesis that conversion of 5-methylcytosine to 5-hydroxymethylcytosine by Tet methylcytosine dioxygenases has a functional role in the demethylation of imprinting control centers in PGCs. Taken together, results from this grant will lead to new insights into the mechanisms that regulate germ line epigenetic inheritance, and in future work our goal will be to prevent epialleles from being acquired and transmitted.